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Lysosomal Dysfunction and Other Pathomechanisms in FTLD: Evidence from Progranulin Genetics and Biology
Abstract
It has been more than a decade since heterozygous loss-of-function mutations in the progranulin gene (GRN) were first identified as an important genetic cause of frontotemporal lobar degeneration (FTLD). Due to the highly diverse biological functions of the progranulin (PGRN) protein, encoded by GRN, multiple possible disease mechanisms have been proposed. Early work focused on the neurotrophic properties of PGRN and its role in the inflammatory response. However, since the discovery of homozygous GRN mutations in patients with a lysosomal storage disorder, investigation into the possible roles of PGRN and its proteolytic cleavage products granulins, in lysosomal function and dysfunction, has taken center stage. In this chapter, we summarize the GRN mutational spectrum and its associated phenotypes followed by an in-depth discussion on the possible disease mechanisms implicated in FTLD-GRN. We conclude with key outstanding questions which urgently require answers to ensure safe and successful therapy development for GRN mutation carriers.
... Notably, despite the reduction of its precursor PGRN due to haploinsufficiency, granulin F levels have been found to be increased in regions of the human FTD-GRN brain [74]. Despite the controversy surrounding the diverse functions and variable expression of individual granulin peptides in pathological states, emerging evidence that links granulin peptides to prion-like TDP-43 cytoplasmic inclusions supports the hypothesis of their potential pathognomonic role in FTD-GRN [68,75,76]. ...
... However, our research did not extend to investigating the impact of individual granulin peptides on TDP-43. Given the conflicting results emerging from various studies regarding individual granulin peptides, there is a clear need to develop antibodies that can specifically detect these peptides and further research into their interactions [74][75][76]. Lastly, although this study focused only on the role of PGRN in human microglia, its effect on interactions with various types of neuronal cells in the brain environment was not determined. Therefore, further studies are necessary to elucidate the impact of PGRN, such as utilization of 3D modeling that incorporates a brain microenvironment with different neuronal cell types, embodying the complexity of a brain's homeostatic and diseased states. ...
Background
Progranulin (PGRN) haploinsufficiency due to progranulin gene (GRN) variants can cause frontotemporal dementia (FTD) with aberrant TAR DNA-binding protein 43 (TDP-43) accumulation. Despite microglial burden with TDP-43-related pathophysiology, direct microglial TDP-43 pathology has not been clarified yet, only emphasized in neuronal pathology. Thus, the objective of this study was to investigate TDP-43 pathology in microglia of patients with PGRN haploinsufficiency.
Methods
To design a human microglial cell model with PGRN haploinsufficiency, monocyte-derived microglia (iMGs) were generated from FTD–GRN patients carrying pathogenic or likely pathogenic variants (p.M1? and p.W147*) and three healthy controls.
Results
iMGs from FTD–GRN patients with PGRN deficiency exhibited severe neuroinflammation phenotype and failure to maintain their homeostatic molecular signatures, along with impaired phagocytosis. In FTD–GRN patients-derived iMGs, significant cytoplasmic TDP-43 aggregation and accumulation of lipid droplets with profound lysosomal abnormalities were observed. These pathomechanisms were mediated by complement C1q activation and upregulation of pro-inflammatory cytokines.
Conclusions
Our study provides considerable cellular and molecular evidence that loss-of-function variants of GRN in human microglia can cause microglial dysfunction with abnormal TDP-43 aggregation induced by inflammatory milieu as well as the impaired lysosome. Elucidating the role of microglial TDP-43 pathology in intensifying neuroinflammation in individuals with FTD due to PGRN deficiency and examining consequential effects on microglial dysfunction might yield novel insights into the mechanisms underlying FTD and neurodegenerative disorders.
... 73,74 In mouse models, TMEM106B deficiency leads to mild myelination deficits, 71,75 microglial survival defects in response to demyelination, 70 reduced survival of Purkinje cells during aging, and mild motor coordination defects. [76][77][78] Since PGRN and TMEM106B regulate different aspects of lysosome functions, 26,27,79 it is not surprising that loss of both PGRN and TMEM106B results in severe lysosomal abnormalities, motor dysfunction, neuronal loss, glial activation, and FTLD-related pathology in mice. 73,80,81 Importantly, these mice exhibit motor defects as early as 2.5 months old and progress with age, resulting in an early death at around 5 months old. ...
... AAV1/9-mediated hPGRN expression partially rescue lysosomal defects in the spinal cord of Tmem106b À/À Grn À/À mice Numerous evidence suggests that both PGRN and TMEM106B play important and non-overlapping roles in proper lysosomal function. 26,27,73,[79][80][81] Deletion of both TMEM106B and PGRN in mice results in severe lysosome abnormalities, including lipofuscin accumulation and upregulation of lysosomal proteins. 73,80,81 We found a significant decrease in lipofuscin signals (Figures 7A and 7B) and levels of lysosome proteases cathepsin D (Cath D) (Figures 7C and 7D) in the spinal cord of Tmem106b À/À Grn À/À mice treated with AAV1-or AAV9-GRN compared to the mice treated with buffer control. ...
Loss of function of progranulin (PGRN), encoded by the granulin (GRN) gene, is implicated in several neurodegenerative diseases. Several therapeutics to boost PGRN levels are currently in clinical trials. However, it is difficult to test the efficacy of PGRN-enhancing drugs in mouse models due to the mild phenotypes of Grn−/− mice. Recently, mice deficient in both PGRN and TMEM106B were shown to develop severe motor deficits and pathology. Here, we show that intracerebral ventricle injection of PGRN-expressing AAV1/9 viruses partially rescues motor deficits, neuronal loss, glial activation, and lysosomal abnormalities in Tmem106b−/−Grn−/− mice. Widespread expression of PGRN is detected in both the brain and spinal cord for both AAV subtypes. However, AAV9 but not AAV1-mediated expression of PGRN results in high levels of PGRN in the serum. Together, these data support using the Tmem106b−/−Grn−/− mouse strain as a robust mouse model to determine the efficacy of PGRN-elevating therapeutics.
... PGRN is a pleiotropic protein and the properties of the full-length protein are distinct from those of granulins. PGRN is considered to be a growth factor, especially for neurons, and important for their survival and outgrowth, whereas some GRNs have inflammatory properties [155]. Progranulin, which is also expressed in microglia, exerts a negative effect on neuroinflammatory processes, such as microgliosis and astrogliosis, and is also involved in repair mechanisms after axonal injury [156]. ...
... Recently it was found that CSF levels of progranulin increase as early as ten years before the clinical presentation of the disease in patients with familial AD; thus, progranulin could represent a possible marker for early prediction of the disease onset. Higher CSF PGRN was linked to more advanced disease stages and cognitive decline in late-onset AD [51,155]. Nevertheless, it is not absolutely specific to AD and has been found to be deregulated in non-AD diseases as well. ...
The accurate diagnosis of neuroinflammatory (NIDs) and neurodegenerative (NDDs) diseases and the stratification of patients into disease subgroups with distinct disease-related characteristics that reflect the underlying pathology represents an unmet clinical need that is of particular interest in the era of emerging disease-modifying therapies (DMT). Proper patient selection for clinical trials and identifying those in the prodromal stages of the diseases or those at high risk will pave the way for precision medicine approaches and halt neuroinflammation and/or neurodegeneration in early stages where this is possible. Towards this direction, novel cerebrospinal fluid (CSF) biomarker candidates were developed to reflect the diseased organ’s pathology better. Μisfolded protein accumulation, microglial activation, synaptic dysfunction, and finally, neuronal death are some of the pathophysiological aspects captured by these biomarkers to support proper diagnosis and screening. We also describe advances in the field of molecular biomarkers, including miRNAs and extracellular nucleic acids known as cell-free DNA and mitochondrial DNA molecules. Here we review the most important of these novel CSF biomarkers of NIDs and NDDs, focusing on their involvement in disease development and emphasizing their ability to define homogeneous disease phenotypes and track potential treatment outcomes that can be mirrored in the CSF compartment.
... While it is not clear where exactly the fibrils reside, within lysosomes or in the cytosol, TMEM106B is localized in lysosomes and is proteolytically processed at the lysosomal membrane defining the lysosome as a central hub. Lysosomes have gradually been recognized as important players in neurodegenerative disorders [3,26,44,55,75], thus it is plausible that the contribution of TMEM106B fibrils to disease might depend on the importance of the lysosome within the specific pathomechanism (Fig. 6). ...
... In fact, homozygous mutations in GRN have been shown to cause a lysosomal storage disorder, neuroid lipofuscinosis (NCL) [61]. PGRN is cleaved within the lysosome into functional granulins and affects several aspects of lysosomal function, including the activity of lysosomal enzymes such as cathepsin D [49,75]. Mutations in GRN result in dysfunctional lysosomes and it is tempting to speculate that loss of PGRN/granulins will directly affect TMEM106B processing or fibril formation by modulating lysosomal health. ...
Since the initial identification of TMEM106B as a risk factor for frontotemporal lobar degeneration (FTLD), multiple genetic studies have found TMEM106B variants to modulate disease risk in a variety of brain disorders and healthy aging. Neurodegenerative disorders are typically characterized by inclusions of misfolded proteins and since lysosomes are an important site for cellular debris clearance, lysosomal dysfunction has been closely linked to neurodegeneration. Consequently, many causal mutations or genetic risk variants implicated in neurodegenerative diseases encode proteins involved in endosomal–lysosomal function. As an integral lysosomal transmembrane protein, TMEM106B regulates several aspects of lysosomal function and multiple studies have shown that proper TMEM106B protein levels are crucial for maintaining lysosomal health. Yet, the precise function of TMEM106B at the lysosomal membrane is undetermined and it remains unclear how TMEM106B modulates disease risk. Unexpectedly, several independent groups recently showed that the C-terminal domain (AA120-254) of TMEM106B forms amyloid fibrils in the brain of patients with a diverse set of neurodegenerative conditions. The recognition that TMEM106B can form amyloid fibrils and is present across neurodegenerative diseases sheds new light on TMEM106B as a central player in neurodegeneration and brain health, but also raises important new questions. In this review, we summarize current knowledge and place a decade’s worth of TMEM106B research into an exciting new perspective.
... Excised granulins were shown to be more stable and to persist in the lysosome long after endocytosis and the disappearance of full-length GRN (Holler et al., 2017). The function of granulins in the lysosome remains to be further elucidated (reviewed in Zhou et al., 2021). ...
... The interaction of GRN with a multitude of extracellular, transmembrane, intracellular, and nuclear proteins is remarkable (De Muynck and Van Damme, 2011;Cui et al., 2019;Zhou et al., 2021) However, the multitude of GRN binding partners is the result of researching GRN biology in diverse histological contexts (peripheral inflammation, vasculogenesis, carcinogenesis and so forth). Regarding GRN biology in the CNS, it seems likely that concerted actions of different binding partners contribute to the neurotrophic effect. ...
Frontotemporal dementia (FTD) is a neurodegenerative disease, leading to behavioral changes and language difficulties. Heterozygous loss-of-function mutations in progranulin (GRN) induce haploinsufficiency of the protein and are associated with up to one-third of all genetic FTD cases worldwide. While the loss of GRN is primarily associated with neurodegeneration, the biological functions of the secreted growth factor-like protein are more diverse, ranging from wound healing, inflammation, vasculogenesis, and metabolic regulation to tumor cell growth and metastasis. To date, no disease-modifying treatments exist for FTD, but different therapeutic approaches to boost GRN levels in the central nervous system are currently being developed (including AAV-mediated GRN gene delivery as well as anti-SORT1 antibody therapy). In this review, we provide an overview of the multifaceted regulation of GRN levels and the corresponding therapeutic avenues. We discuss the opportunities, advantages, and potential drawbacks of the diverse approaches. Additionally, we highlight the therapeutic potential of elevating GRN levels beyond patients with loss-of-function mutations in GRN.
... Given that GRN-linked frontotemporal dementia arises from progranulin haploinsufficiency, increasing the expression of progranulin from a remaining WT copy of the GRN gene, gene therapy to boost GRN expression, and protein replacement therapy are all actively investigated therapeutic strategies (Zhou et al., 2021;Logan et al., 2021). However, the success of such approaches depends on a detailed understanding of the mechanisms that either stimulate the activity of progranulin/granulins within lysosomes or ensure the delivery of progranulin to its site of action within the lumen of lysosomes. ...
Progranulin is a lysosomal protein whose haploinsufficiency causes frontotemporal dementia, while homozygous loss of progranulin causes neuronal ceroid lipofuscinosis, a lysosomal storage disease. The sensitivity of cells to progranulin deficiency raises important questions about how cells coordinate intracellular trafficking of progranulin to ensure its efficient delivery to lysosomes. In this study, we discover that progranulin interactions with prosaposin, another lysosomal protein, first occur within the lumen of the endoplasmic reticulum (ER) and are required for the efficient ER exit of progranulin. Mechanistically, we identify an interaction between prosaposin and Surf4, a receptor that promotes loading of lumenal cargos into COPII-coated vesicles, and establish that Surf4 is critical for the efficient export of progranulin and prosaposin from the ER. Collectively, this work demonstrates that a network of interactions occurring early in the secretory pathway promote the ER exit and subsequent lysosomal delivery of newly translated progranulin and prosaposin.
Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin ( Grn ), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome‐related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes.
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (i.e. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (i.e. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (i.e. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (i.e. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (i.e. APOE, PSEN1, APP) and Parkinson's (i.e. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.
Progranulin is a lysosomal protein whose haploinsufficiency causes frontotemporal dementia while homozygous loss of progranulin causes neuronal ceroid lipofuscinosis, a lysosomal storage disease. The sensitivity of cells to progranulin deficiency raises important questions about how cells coordinate intracellular trafficking of progranulin to ensure its efficient delivery to lysosomes. In this study, we discover that progranulin interacts with prosaposin, another lysosomal protein, within the lumen of the endoplasmic reticulum (ER) and that prosaposin is required for the efficient ER exit of progranulin. Mechanistically, we identify an interaction between prosaposin and Surf4, a receptor that promotes loading of lumenal cargos into COPII coated vesicles, and establish that Surf4 is critical for the efficient export of progranulin and prosaposin from the ER. Collectively, this work demonstrates a network of interactions occurring early in the secretory pathway that promote the ER exit and subsequent lysosomal delivery of newly translated progranulin and prosaposin.
Purpose:
The authors previously reported that progranulin attenuated retinal degeneration. The present study focused on the role of progranulin and its cleavage products, granulins, in the pathogenesis of photoreceptor degeneration.
Methods:
Photoreceptor degeneration was induced with excessive exposure of murine photoreceptor cells and the retinas of albino mice to white fluorescent light. Damaged photoreceptor cells and retinas were examined using a cell death assay, western blotting, and immunostaining.
Results:
Even after proteolytic cleavage, treatment with progranulin or its cleavage products or both exerted protective effects on photoreceptors against light exposure. In the murine retina, the expression levels of granulins and the macrophage and microglia marker Iba-1 were increased at 48 h after light exposure. Additionally, progranulin+ and Iba-1+ double-positive cells had accumulated in the outer nuclear layer, the primary location of photoreceptor cells.
Conclusions:
These results suggest that progranulin or its cleavage products, granulins, or both may be therapeutic targets for age-related macular degeneration and other neurodegenerative diseases.
Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only β-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature β-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated β-glucocerebrosidase. Immunostaining revealed loss of neuronal β-glucocerebrosidase in FTD-GRN patients. To investigate the effects of progranulin insufficiency on β-glucocerebrosidase outside of the context of neurodegeneration, we investigated β-glucocerebrosidase activity in progranulin-insufficient mice. Brains from Grn-/- mice had lower β-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs β-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired β-glucocerebrosidase processing.
Abstract Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer’s disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aβ) plaques, while PSAP expression was mainly detected in neurons and Aβ plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aβ), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aβ plaques did not. Aβ plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aβ plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.
Homozygous mutations in the progranulin gene (GRN) are associated with neuronal ceroid lipofuscinosis 11 (CLN11), a rare lysosomal-storage disorder characterized by cerebellar ataxia, seizures, retinitis pigmentosa, and cognitive disorders, usually beginning between 13 and 25 years of age. This is a rare condition, previously reported in only four families. In contrast, heterozygous GRN mutations are a major cause of frontotemporal dementia associated with neuronal cytoplasmic TDP-43 inclusions. We identified homozygous GRN mutations in six new patients. The phenotypic spectrum is much broader than previously reported, with two remarkably distinct presentations, depending on the age of onset. A childhood/juvenile form is characterized by classical CLN11 symptoms at an early age at onset. Unexpectedly, other homozygous patients presented a distinct delayed phenotype of frontotemporal dementia and parkinsonism after 50 years; none had epilepsy or cerebellar ataxia. Another major finding of this study is that all GRN mutations may not have the same impact on progranulin protein synthesis. A hypomorphic effect of some mutations is supported by the presence of residual levels of plasma progranulin and low levels of normal transcript detected in one case with a homozygous splice-site mutation and late onset frontotemporal dementia. This is a new critical finding that must be considered in therapeutic trials based on replacement strategies. The first neuropathological study in a homozygous carrier provides new insights into the pathological mechanisms of the disease. Hallmarks of neuronal ceroid lipofuscinosis were present. The absence of TDP-43 cytoplasmic inclusions markedly differs from observations of heterozygous mutations, suggesting a pathological shift between lysosomal and TDP-43 pathologies depending on the mono or bi-allelic status. An intriguing observation was the loss of normal TDP-43 staining in the nucleus of some neurons, which could be the first stage of the TDP-43 pathological process preceding the formation of typical cytoplasmic inclusions. Finally, this study has important implications for genetic counselling and molecular diagnosis. Semi-dominant inheritance of GRN mutations implies that specific genetic counselling should be delivered to children and parents of CLN11 patients, as they are heterozygous carriers with a high risk of developing dementia. More broadly, this study illustrates the fact that genetic variants can lead to different phenotypes according to their mono- or bi-allelic state, which is a challenge for genetic diagnosis.
Type I interferons (IFNs) play a critical role in host defense against influenza virus infection, and the mechanism of influenza virus to evade type I IFNs responses remains to be fully understood. Here, we found that progranulin (PGRN) was significantly increased both in vitro and in vivo during influenza virus infection. Using a PGRN knockdown assay and PGRN-deficient mice model, we demonstrated that influenza virus-inducing PGRN negatively regulated type I IFNs production by inhibiting the activation of NF-κB and IRF3 signaling. Furthermore, we showed that PGRN directly interacted with NF-κB essential modulator (NEMO) via its Grn CDE domains. We also verified that PGRN recruited A20 to deubiquitinate K63-linked polyubiquitin chains on NEMO at K264. In addition, we found that macrophage played a major source of PGRN during influenza virus infection, and PGRN neutralizing antibodies could protect against influenza virus-induced lethality in mice. Our data identify a PGRN-mediated IFN evasion pathway exploited by influenza virus with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of PGRN in innate immunity.
Mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, are among the most common known genetic risk factors for the development of Parkinson disease and related synucleinopathies. A great deal is known about GBA1, as mutations in GBA1 are causal for the rare autosomal storage disorder Gaucher disease. Over the past decades, significant progress has been made in understanding the genetics and cell biology of glucocerebrosidase. A least 495 different mutations, found throughout the 11 exons of the gene are reported, including both common and rare variants. Mutations in GBA1 may lead to degradation of the protein, disruptions in lysosomal targeting and diminished performance of the enzyme in the lysosome.Gaucher disease is phenotypically diverse and has both neuronopathic and non-neuronopathic forms. Both patients with Gaucher disease and heterozygous carriers are at increased risk of developing Parkinson disease and Dementia with Lewy Bodies, although our understanding of the mechanism for this association remains incomplete. There appears to be an inverse relationship between glucocerebrosidase and α-synuclein levels, and even patients with sporadic Parkinson disease have decreased glucocerebrosidase. Glucocerebrosidase may interact with α-synuclein to maintain basic cellular functions, or impaired glucocerebrosidase could contribute to Parkinson pathogenesis by disrupting lysosomal homeostasis, enhancing endoplasmic reticulum stress or contributing to mitochondrial impairment. However, the majority of patients with GBA1 mutations never develop parkinsonism, so clearly other risk factors play a role. Treatments for Gaucher disease have been developed that increase visceral glucocerebrosidase levels and decrease lipid storage, although they have yet to properly address the neurological defects associated with impaired glucocerebrosidase. Mouse and induced pluripotent stem cell derived models have improved our understanding of glucocerebrosidase function and the consequences of its deficiency. These models have been used to test novel therapies including chaperone proteins, histone deacetylase inhibitors, and gene therapy approaches that enhance glucocerebrosidase levels and could prove efficacious in the treatment of forms of parkinsonism. Consequently, this rare monogenic disorder, Gaucher disease, provides unique insights directly applicable to our understanding and treatment of Parkinson disease, a common and complex neurodegenerative disorder.
Limbic‐predominant age‐related TAR‐DNA‐binding protein‐43 (TDP‐43) encephalopathy with hippocampal sclerosis pathology (LATE‐NC+HS) is a neurodegenerative disorder characterized by severe hippocampal CA1 neuron loss and TDP‐43‐pathology, leading to cognitive dysfunction and dementia. Polymorphisms in GRN, TMEM106B and ABCC9 are proposed as LATE‐NC+HS risk factors in brain bank collections. To replicate these results in independent population‐representative cohorts, hippocampal sections from brains donated to three such studies (Cambridge City over 75‐Cohort [CC75C], Cognitive Function and Ageing Study [CFAS], and Vantaa 85+ Study) were stained with haematoxylin‐eosin (n=744) and anti‐pTDP‐43 (n=713), and evaluated for LATE‐NC+HS and TDP‐43 pathology. Single nucleotide polymorphism genotypes in GRN rs5848, TMEM106B rs1990622 and ABCC9 rs704178 were determined. LATE‐NC+HS (n=58) was significantly associated with the GRN rs5848 genotype (χ2(2)=20.61, p<0.001) and T‐allele (χ2(1)=21.04, p<0.001), and TMEM106B rs1990622 genotype (Fisher′s exact test, p<0.001) and A‐allele (χ2(1)=25.75, p<0.001). No differences in ABCC9 rs704178 genotype or allele frequency were found between LATE‐NC+HS and non‐ LATE‐NC+HS neuropathology cases. Dentate gyrus TDP‐43 pathology associated with GRN and TMEM106B variations, but the association with TMEM106B nullified when LATE‐NC+HS cases were excluded. Our results indicate that GRN and TMEM106B are associated with severe loss of CA1 neurons in the aging brain, while ABCC9 was not confirmed as a genetic risk factor for LATE‐NC+HS. The association between TMEM106B and LATE‐NC+HS may be independent of dentate TDP‐43 pathology. This article is protected by copyright. All rights reserved.
Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GCase), and is essential for proper GCase activity. GCase activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.
Abstract Microglia adopt numerous fates with homeostatic microglia (HM) and a microglial neurodegenerative phenotype (MGnD) representing two opposite ends. A number of variants in genes selectively expressed in microglia are associated with an increased risk for neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). Among these genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). Both cause neurodegeneration by mechanisms involving loss of function. We have now isolated microglia from Grn−/− mice and compared their transcriptomes to those of Trem2−/− mice. Surprisingly, while loss of Trem2 enhances the expression of genes associated with a homeostatic state, microglia derived from Grn−/− mice showed a reciprocal activation of the MGnD molecular signature and suppression of gene characteristic for HM. The opposite mRNA expression profiles are associated with divergent functional phenotypes. Although loss of TREM2 and progranulin resulted in opposite activation states and functional phenotypes of microglia, FDG (fluoro‐2‐deoxy‐d‐glucose)‐μPET of brain revealed reduced glucose metabolism in both conditions, suggesting that opposite microglial phenotypes result in similar wide spread brain dysfunction.
Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) represents the most common pathological subtype of FTLD. We established the international FTLD-TDP whole-genome sequencing consortium to thoroughly characterize the known genetic causes of FTLD-TDP and identify novel genetic risk factors. Through the study of 1131 unrelated Caucasian patients, we estimated that C9orf72 repeat expansions and GRN loss-of-function mutations account for 25.5% and 13.9% of FTLD-TDP patients, respectively. Mutations in TBK1 (1.5%) and other known FTLD genes (1.4%) were rare, and the disease in 57.7% of FTLD-TDP patients was unexplained by the known FTLD genes. To unravel the contribution of common genetic factors to the FTLD-TDP etiology in these patients, we conducted a two-stage association study comprising the analysis of whole-genome sequencing data from 517 FTLD-TDP patients and 838 controls, followed by targeted genotyping of the most associated genomic loci in 119 additional FTLD-TDP patients and 1653 controls. We identified three genome-wide significant FTLD-TDP risk loci: one new locus at chromosome 7q36 within the DPP6 gene led by rs118113626 (p value = 4.82e − 08, OR = 2.12), and two known loci: UNC13A, led by rs1297319 (p value = 1.27e − 08, OR = 1.50) and HLA-DQA2 led by rs17219281 (p value = 3.22e − 08, OR = 1.98). While HLA represents a locus previously implicated in clinical FTLD and related neurodegenerative disorders, the association signal in our study is independent from previously reported associations. Through inspection of our whole-genome sequence data for genes with an excess of rare loss-of-function variants in FTLD-TDP patients (n ≥ 3) as compared to controls (n = 0), we further discovered a possible role for genes functioning within the TBK1-related immune pathway (e.g., DHX58, TRIM21, IRF7) in the genetic etiology of FTLD-TDP. Together, our study based on the largest cohort of unrelated FTLD-TDP patients assembled to date provides a comprehensive view of the genetic landscape of FTLD-TDP, nominates novel FTLD-TDP risk loci, and strongly implicates the immune pathway in FTLD-TDP pathogenesis.
Progranulin (PGRN) is predominantly expressed by microglia in the brain, and genetic and experimental evidence suggests a critical role in Alzheimer's disease (AD). We asked whether PGRN expression is changed in a disease severity-specific manner in AD. We measured PGRN in cerebrospinal fluid (CSF) in two of the best-characterized AD patient cohorts, namely the Dominant Inherited Alzheimer's Disease Network (DIAN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). In carriers of AD causing dominant mutations, cross-sectionally assessed CSF PGRN increased over the course of the disease and significantly differed from non-carriers 10 years before the expected symptom onset. In late-onset AD, higher CSF PGRN was associated with more advanced disease stages and cognitive impairment. Higher CSF PGRN was associated with higher CSF soluble TREM2 (triggering receptor expressed on myeloid cells 2) only when there was underlying pathology, but not in controls. In conclusion, we demonstrate that, although CSF PGRN is not a diagnostic biomarker for AD, it may together with sTREM2 reflect microglial activation during the disease. © 2018 The Authors. Published under the terms of the CC BY 4.0 license
Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.
Tay-Sachs disease (TSD) is a lethal lysosomal storage disease (LSD) caused by mutations in the HexA gene, which can lead to deficiency of β-hexosaminidase A (HexA) activity and consequent accumulation of its substrate, GM2 ganglioside. Recent reports that progranulin (PGRN) functions as a chaperone of lysosomal enzymes and its deficiency is associated with LSDs, including Gaucher disease and neuronal ceroid lipofuscinosis, prompted us to screen the effects of recombinant PGRN on lysosomal storage in fibroblasts from 11 patients affected by various LSDs, which led to the isolation of TSD in which PGRN demonstrated the best effects in reducing lysosomal storage. Subsequent in vivo studies revealed significant GM2 accumulation and the existence of typical TSD cells containing zebra bodies in both aged and ovalbumin-challenged adult PGRN-deficient mice. In addition, HexA, but not HexB, was aggregated in PGRN-deficient cells. Furthermore, recombinant PGRN significantly reduced GM2 accumulation and lysosomal storage in these animal models. Mechanistic studies indicated that PGRN bound to HexA through granulins G and E domain and increased the enzymatic activity and lysosomal delivery of HexA. More importantly, Pcgin, an engineered PGRN derivative bearing the granulin E domain, also effectively bound to HexA and reduced the GM2 accumulation. Collectively, these studies not only provide new insights into the pathogenesis of TSD but may also have implications for developing PGRN-based therapy for this life-threatening disorder.
Key messages
• GM2 accumulation and the existence of typical TSD cells containing zebra bodies are detected in both aged and ovalbumin-challenged adult PGRN deficient mice.
• Recombinant PGRN significantly reduces GM2 accumulation and lysosomal storage both in vivo and in vitro, which works through increasing the expression and lysosomal delivery of HexA.
• Pcgin, an engineered PGRN derivative bearing the granulin E domain, also effectively binds to to HexA and reduces GM2 accumulation.
Background:
Heterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear.
Methods:
mRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins.
Results:
We demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN.
Conclusions:
The present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells.
Background
Loss of function mutations in progranulin (GRN) are a major cause of frontotemporal dementia (FTD). Progranulin is a secreted glycoprotein that localizes to lysosomes and is critical for proper lysosomal function. Heterozygous GRN mutation carriers develop FTD with TDP-43 pathology and exhibit signs of lysosomal dysfunction in the brain, with increased levels of lysosomal proteins and lipofuscin accumulation. Homozygous GRN mutation carriers develop neuronal ceroid lipofuscinosis (NCL), an earlier-onset lysosomal storage disorder caused by severe lysosomal dysfunction. Multiple genome-wide association studies have shown that risk of FTD in GRN mutation carriers is modified by polymorphisms in TMEM106B, which encodes a lysosomal membrane protein. Risk alleles of TMEM106B may increase TMEM106B levels through a variety of mechanisms. Brains from FTD patients with GRN mutations exhibit increased TMEM106B expression, and protective TMEM106B polymorphisms are associated with decreased TMEM106B expression. Together, these data raise the possibility that reduction of TMEM106B levels may protect against the pathogenic effects of progranulin haploinsufficiency. Methods
We crossed Tmem106b +/− mice with Grn +/− mice, which model the progranulin haploinsufficiency of GRN mutation carriers and develop age-dependent social deficits and lysosomal abnormalities in the brain. We tested whether partial Tmem106b reduction could normalize the social deficits and lysosomal abnormalities of Grn +/− mice. ResultsPartial reduction of Tmem106b levels did not correct the social deficits of Grn +/− mice. Tmem106b reduction also failed to normalize most lysosomal abnormalities of Grn +/− mice, except for β-glucuronidase activity, which was suppressed by Tmem106b reduction and increased by progranulin insufficiency. Conclusions
These data do not support the hypothesis that Tmem106b reduction protects against the pathogenic effects of progranulin haploinsufficiency, but do show that Tmem106b reduction normalizes some lysosomal phenotypes in Grn +/− mice.
Cells have evolved quality control pathways to prevent the accumulation of improperly localized proteins, which are often toxic. One of these pathways, regulation of aberrant protein production (RAPP), recognizes aberrant secretory proteins during translation and degrades the associated mRNA. Here, we demonstrate endogenous RAPP substrates. Haploinsufficiency of the secretory protein progranulin (GRN) is associated with the neurodegenerative disease frontotemporal lobar degeneration (FTLD). Our results show FTLD-associated GRN mutations W7R and A9D disrupt co-translational interaction with a targeting factor, signal recognition particle (SRP). This triggers RAPP and initiates specific mRNA degradation. Conversely, wild-type GRN and the naturally occurring polymorphism V5L GRN are efficiently expressed and secreted. Thus, RAPP plays a role in the molecular pathology of A9D GRN and W7R GRN.
Loss-of-function mutations in progranulin (GRN) and a non-coding (GGGGCC)n hexanucleotide repeat expansions in C9ORF72 are the two most common genetic causes of frontotemporal lobar degeneration with aggregates of TAR DNA binding protein 43 (FTLD-TDP). TMEM106B encodes a type II transmembrane protein with unknown function. Genetic variants in TMEM106B associated with reduced TMEM106B levels have been identified as disease modifiers in individuals with GRN mutations and C9ORF72 expansions. Recently, loss of Tmem106b has been reported to protect the FTLD-like phenotypes in Grn−/− mice. Here, we generated Tmem106b−/− mice and examined whether loss of Tmem106b could rescue FTLD-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity. Our results showed that neither partial nor complete loss of Tmem106b was able to rescue behavioral deficits induced by the expression of (GGGGCC)66 repeats (66R). Loss of Tmem106b also failed to ameliorate 66R-induced RNA foci, dipeptide repeat protein formation and pTDP-43 pathological burden. We further found that complete loss of Tmem106b increased astrogliosis, even in the absence of 66R, and failed to rescue 66R-induced neuronal cell loss, whereas partial loss of Tmem106b significantly rescued the neuronal cell loss but not neuroinflammation induced by 66R. Finally, we showed that overexpression of 66R did not alter expression of Tmem106b and other lysosomal genes in vivo, and subsequent analyses in vitro found that transiently knocking down C9ORF72, but not overexpression of 66R, significantly increased TMEM106B and other lysosomal proteins. In summary, reducing Tmem106b levels failed to rescue FTLD-like phenotypes in a mouse model mimicking the toxic gain-of-functions associated with overexpression of 66R. Combined with the observation that loss of C9ORF72 and not 66R overexpression was associated with increased levels of TMEM106B, this work suggests that the protective TMEM106B haplotype may exert its effect in expansion carriers by counteracting lysosomal dysfunction resulting from a loss of C9ORF72.
Electronic supplementary material
The online version of this article (10.1186/s40478-018-0545-x) contains supplementary material, which is available to authorized users.
Progranulin (PGRN), encoded by the GRN gene in humans, is a secreted growth factor implicated in a multitude of processes ranging from regulation of inflammation to wound healing and tumorigenesis. The clinical importance of PGRN became especially evident in 2006, when heterozygous mutations in the GRN gene, resulting in haploinsufficiency, were found to be one of the main causes of frontotemporal lobar degeneration (FTLD). FTLD is a clinically heterogenous disease that results in the progressive atrophy of the frontal and temporal lobes of the brain. Despite significant research, the exact function of PGRN and its mechanistic relationship to FTLD remain unclear. However, growing evidence suggests a role for PGRN in the lysosome—most striking being that homozygous GRN mutation leads to neuronal ceroid lipofuscinosis, a lysosomal storage disease. Since this discovery, several links between PGRN and the lysosome have been established, including the existence of two independent lysosomal trafficking pathways, intralysosomal processing of PGRN into discrete functional peptides, and direct and indirect regulation of lysosomal hydrolases. Here, we summarize the cellular functions of PGRN, its roles in the nervous system, and its link to multiple neurodegenerative diseases, with a particular focus dedicated to recent lysosome-related mechanistic developments.
Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.
Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result fromGRNnonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterizedGrn
R493X
knockin mice, which model the most common humanGRNmutation, a premature stop codon at arginine 493 (R493X). HomozygousGrn
R493X
mice have markedly reducedGrnmRNA levels, lack detectable progranulin protein, and phenocopyGrnknockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels inGrn
R493X
mice and cell lines and in fibroblasts from patients containing theGRN
R493X
mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation.
Mutations in the progranulin (PGRN) gene cause a tau pathology-negative and TDP43 pathology-positive form of frontotemporal lobar degeneration (FTLD-TDP). We generated a knock-in mouse harboring the R504X mutation (PGRN-KI). Phosphoproteomic analysis of this model revealed activation of signaling pathways connecting PKC and MAPK to tau prior to TDP43 aggregation and cognitive impairments, and identified PKCα as the kinase responsible for the early-stage tau phosphorylation at Ser203. Disinhibition of Gas6 binding to Tyro3 due to PGRN reduction results in activation of PKCα via PLCγ, inducing tau phosphorylation at Ser203, mislocalization of tau to dendritic spines, and spine loss. Administration of a PKC inhibitor, B-Raf inhibitor, or knockdown of molecules in the Gas6-Tyro3-tau axis rescues spine loss and cognitive impairment of PGRN-KI mice. Collectively, these results suggest that targeting of early-stage and aggregation-independent tau signaling represents a promising therapeutic strategy for this disease.
We investigated whether progranulin plasma levels are predictors of the presence of progranulin gene (GRN) null mutations or of the development of symptoms in asymptomatic at risk members participating in the Genetic Frontotemporal Dementia Initiative (GENFI), including 19 patients, 64 asymptomatic carriers and 77 non-carriers. In addition, we evaluated a possible role of TMEM106B rs1990622 as a genetic modifier and correlated progranulin plasma levels and gray matter atrophy. Plasma progranulin mean±SD plasma levels in patients and asymptomatic carriers were significantly decreased compared with non-carriers (30.5±13.0 and 27.7±7.5 versus 99.6±24.8 ng/ml, P<0.00001). Considering the threshold of >61.55 ng/ml, the test had a sensitivity of 98.8% and a specificity of 97.5% in predicting the presence of a mutation, independent of symptoms. No correlations were found between progranulin plasma levels and age, years from average age at onset in each family, or TMEM106B rs1990622 genotype (P>0.05). Plasma progranulin levels did not correlate with brain atrophy. Plasma progranulin levels predict the presence of GRN null mutations independent of proximity to symptoms and brain atrophy.
Progranulin, a secreted glycoprotein, is encoded in humans by the single GRN gene. Progranulin consists of seven and a half, tandemly repeated, non-identical copies of the 12 cysteine granulin motif. Many cellular processes and diseases are associated with this unique pleiotropic factor that include, but are not limited to, embryogenesis, tumorigenesis, inflammation, wound repair, neurodegeneration and lysosome function. Haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to frontotemporal lobar degeneration, a progressive neuronal atrophy that presents in patients as frontotemporal dementia. Frontotemporal dementia is an early onset form of dementia, distinct from Alzheimer's disease. The GRN-related form of frontotemporal lobar dementia is a proteinopathy characterized by the appearance of neuronal inclusions containing ubiquitinated and fragmented TDP-43 (encoded by TARDBP). The neurotrophic and neuro-immunomodulatory properties of progranulin have recently been reported but are still not well understood. Gene delivery of GRN in experimental models of Alzheimer's- and Parkinson's-like diseases inhibits phenotype progression. Here we review what is currently known concerning the molecular function and mechanism of action of progranulin in normal physiological and pathophysiological conditions in both in vitro and in vivo models. The potential therapeutic applications of progranulin in treating neurodegenerative diseases are highlighted.
Background
Mutations resulting in progranulin (PGRN) haploinsufficiency cause frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), a devastating neurodegenerative disease. PGRN is localized to the lysosome and important for proper lysosome function. However, the metabolism of PGRN in the lysosome is still unclear.
Results
Here, we report that PGRN is processed into ~10 kDa peptides intracellularly in multiple cell types and tissues and this processing is dependent on lysosomal activities. PGRN endocytosed from the extracellular space is also processed in a similar manner. We further demonstrated that multiple cathepsins are involved in PGRN processing and cathepsin L cleaves PGRN in vitro.
Conclusions
Our data support that PGRN is processed in the lysosome through the actions of cathepsins.
Homozygous or heterozygous mutations in the GRN gene, encoding progranulin (PGRN), cause neuronal ceroid lipofuscinosis (NCL) or frontotemporal dementia (FTD), respectively. NCL and FTD are characterized by lysosome dysfunction and neurodegeneration, indicating PGRN is important for lysosome homeostasis in the brain. PGRN is trafficked to the lysosome where its functional role is unknown. PGRN can be cleaved into seven 6-kDa proteins called granulins (GRNs); however, little is known about how GRNs are produced or if levels of GRNs are altered in FTD-GRN mutation carriers. Here, we report the identification and characterization of antibodies that reliably detect several human GRNs by immunoblot and immunocytochemistry. Using these tools, we find that endogenous GRNs are present within multiple cell lines and are constitutively produced. Further, extracellular PGRN is endocytosed and rapidly processed into stable GRNs within lysosomes. Processing of PGRN into GRNs is conserved between humans and mice and is modulated by sortilin expression and mediated by cysteine proteases (i.e. cathpesin L). Induced lysosome dysfunction caused by alkalizing agents or increased expression of transmembrane protein 106B (TMEM106B) inhibit processing of PGRN into GRNs. Finally, we find that multiple GRNs are haploinsufficient in primary fibroblasts and cortical brain tissue from FTD-GRN patients. Taken together, our findings raise the interesting possibility that GRNs carry out critical lysosomal functions and that loss of GRNs should be explored as an initiating factor in lysosomal dysfunction and neurodegeneration caused by GRN mutations.
Loss-of-function mutations in GRN cause frontotemporal dementia (FTD) with transactive response DNA-binding protein of 43 kD (TDP-43)–positive inclusions and neuronal ceroid lipofuscinosis (NCL). There are no disease-modifying therapies for either FTD or NCL, in part because of a poor understanding of how mutations in genes such as GRN contribute to disease pathogenesis and neurodegeneration. By studying mice lacking progranulin (PGRN), the protein encoded by GRN , we discovered multiple lines of evidence that PGRN deficiency results in impairment of autophagy, a key cellular degradation pathway. PGRN-deficient mice are sensitive to Listeria monocytogenes because of deficits in xenophagy, a specialized form of autophagy that mediates clearance of intracellular pathogens. Cells lacking PGRN display reduced autophagic flux, and pathological forms of TDP-43 typically cleared by autophagy accumulate more rapidly in PGRN-deficient neurons. Our findings implicate autophagy as a novel therapeutic target for GRN -associated NCL and FTD and highlight the emerging theme of defective autophagy in the broader FTD/amyotrophic lateral sclerosis spectrum of neurodegenerative disease.
Haploinsufficiency of GRN, the gene encoding progranulin (PGRN), causes frontotemporal lobar degeneration (FTLD), the second most common cause of early-onset dementia. Receptor-mediated lysosomal targeting has been shown to regulate brain PGRN levels, and complete deficiency of PGRN is a direct cause of neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Here we show that the lysosomal cysteine protease cathepsin L (Cat L) can mediate the proteolytic cleavage of intracellular PGRN into poly-granulin and granulin fragments. Further, PGRN and Cat L co-localize in lysosomes of HEK293 cells, iPSC-derived neurons and human cortical neurons from human postmortem tissue. These data identify Cat L as a key intracellular lysosomal PGRN protease, and provides an intriguing new link between lysosomal dysfunction and FTLD.
Electronic supplementary material
The online version of this article (doi:10.1186/s13024-017-0196-6) contains supplementary material, which is available to authorized users.
Haploinsufficiency of progranulin (PGRN) due to mutations in the granulin (GRN) gene causes frontotemporal lobar degeneration (FTLD), and complete loss of PGRN leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests that PGRN is essential for proper lysosomal function, but the precise mechanisms involved are not known. Here, we show that PGRN facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. We found reduced levels of PSAP in neurons both in mice deficient in PGRN and in human samples from FTLD patients due to GRN mutations. Furthermore, mice with reduced PSAP expression demonstrated FTLD-like pathology and behavioural changes. Thus, our data demonstrate a role of PGRN in PSAP lysosomal trafficking and suggest that impaired lysosomal trafficking of PSAP is an underlying disease mechanism for NCL and FTLD due to GRN mutations.
A blood cell protein-expression atlas
Genome-wide analyses are increasingly providing resources for advances in basic and applied biomedical science. Uhlen et al. performed a global expression analysis of human blood cell types and integrated this data with data across all major human tissues and organs in the human protein atlas. This comprehensive compendium allows for classification of all human protein-coding genes with regard to their tissue- and cell-type distribution.
Science , this issue p. eaax9198
Background Frontotemporal dementia is a heterogenous neurodegenerative disorder, with about a third of cases being genetic. Most of this genetic component is accounted for by mutations in GRN, MAPT, and C9orf72. In this study, we aimed to complement previous phenotypic studies by doing an international study of age at symptom onset, age at death, and disease duration in individuals with mutations in GRN, MAPT, and C9orf72. Methods In this international, retrospective cohort study, we collected data on age at symptom onset, age at death, and disease duration for patients with pathogenic mutations in the GRN and MAPT genes and pathological expansions in the C9orf72 gene through the Frontotemporal Dementia Prevention Initiative and from published papers. We used mixed effects models to explore differences in age at onset, age at death, and disease duration between genetic groups and individual mutations. We also assessed correlations between the age at onset and at death of each individual and the age at onset and at death of their parents and the mean age at onset and at death of their family members. Lastly, we used mixed effects models to investigate the extent to which variability in age at onset and at death could be accounted for by family membership and the specific mutation carried. Findings Data were available from 3403 individuals from 1492 families: 1433 with C9orf72 expansions (755 families), 1179 with GRN mutations (483 families, 130 different mutations), and 791 with MAPT mutations (254 families, 67 different mutations). Mean age at symptom onset and at death was 49·5 years (SD 10·0; onset) and 58·5 years (11·3; death) in the MAPT group, 58·2 years (9·8; onset) and 65·3 years (10·9; death) in the C9orf72 group, and 61·3 years (8·8; onset) and 68·8 years (9·7; death) in the GRN group. Mean disease duration was 6·4 years (SD 4·9) in the C9orf72 group, 7·1 years (3·9) in the GRN group, and 9·3 years (6·4) in the MAPT group. Individual age at onset and at death was significantly correlated with both parental age at onset and at death and with mean family age at onset and at death in all three groups, with a stronger correlation observed in the MAPT group (r=0·45 between individual and parental age at onset, r=0·63 between individual and mean family age at onset, r=0·58 between individual and parental age at death, and r=0·69 between individual and mean family age at death) than in either the C9orf72 group (r=0·32 individual and parental age at onset, r=0·36 individual and mean family age at onset, r=0·38 individual and parental age at death, and r=0·40 individual and mean family age at death) or the GRN group (r=0·22 individual and parental age at onset, r=0·18 individual and mean family age at onset, r=0·22 individual and parental age at death, and r=0·32 individual and mean family age at death). Modelling showed that the variability in age at onset and at death in the MAPT group was explained partly by the specific mutation (48%, 95% CI 35–62, for age at onset; 61%, 47–73, for age at death), and even more by family membership (66%, 56–75, for age at onset; 74%, 65–82, for age at death). In the GRN group, only 2% (0–10) of the variability of age at onset and 9% (3–21) of that of age of death was explained by the specific mutation, whereas 14% (9–22) of the variability of age at onset and 20% (12–30) of that of age at death was explained by family membership. In the C9orf72 group, family membership explained 17% (11–26) of the variability of age at onset and 19% (12–29) of that of age at death. Interpretation Our study showed that age at symptom onset and at death of people with genetic frontotemporal dementia is influenced by genetic group and, particularly for MAPT mutations, by the specific mutation carried and by family membership. Although estimation of age at onset will be an important factor in future pre-symptomatic therapeutic trials for all three genetic groups, our study suggests that data from other members of the family will be particularly helpful only for individuals with MAPT mutations. Further work in identifying both genetic and environmental factors that modify phenotype in all groups will be important to improve such estimates. Funding UK Medical Research Council, National Institute for Health Research, and Alzheimer's Society.
Background:
Frontotemporal dementia is a heterogenous neurodegenerative disorder, with about a third of cases being genetic. Most of this genetic component is accounted for by mutations in GRN, MAPT, and C9orf72. In this study, we aimed to complement previous phenotypic studies by doing an international study of age at symptom onset, age at death, and disease duration in individuals with mutations in GRN, MAPT, and C9orf72.
Methods:
In this international, retrospective cohort study, we collected data on age at symptom onset, age at death, and disease duration for patients with pathogenic mutations in the GRN and MAPT genes and pathological expansions in the C9orf72 gene through the Frontotemporal Dementia Prevention Initiative and from published papers. We used mixed effects models to explore differences in age at onset, age at death, and disease duration between genetic groups and individual mutations. We also assessed correlations between the age at onset and at death of each individual and the age at onset and at death of their parents and the mean age at onset and at death of their family members. Lastly, we used mixed effects models to investigate the extent to which variability in age at onset and at death could be accounted for by family membership and the specific mutation carried.
Findings:
Data were available from 3403 individuals from 1492 families: 1433 with C9orf72 expansions (755 families), 1179 with GRN mutations (483 families, 130 different mutations), and 791 with MAPT mutations (254 families, 67 different mutations). Mean age at symptom onset and at death was 49·5 years (SD 10·0; onset) and 58·5 years (11·3; death) in the MAPT group, 58·2 years (9·8; onset) and 65·3 years (10·9; death) in the C9orf72 group, and 61·3 years (8·8; onset) and 68·8 years (9·7; death) in the GRN group. Mean disease duration was 6·4 years (SD 4·9) in the C9orf72 group, 7·1 years (3·9) in the GRN group, and 9·3 years (6·4) in the MAPT group. Individual age at onset and at death was significantly correlated with both parental age at onset and at death and with mean family age at onset and at death in all three groups, with a stronger correlation observed in the MAPT group (r=0·45 between individual and parental age at onset, r=0·63 between individual and mean family age at onset, r=0·58 between individual and parental age at death, and r=0·69 between individual and mean family age at death) than in either the C9orf72 group (r=0·32 individual and parental age at onset, r=0·36 individual and mean family age at onset, r=0·38 individual and parental age at death, and r=0·40 individual and mean family age at death) or the GRN group (r=0·22 individual and parental age at onset, r=0·18 individual and mean family age at onset, r=0·22 individual and parental age at death, and r=0·32 individual and mean family age at death). Modelling showed that the variability in age at onset and at death in the MAPT group was explained partly by the specific mutation (48%, 95% CI 35-62, for age at onset; 61%, 47-73, for age at death), and even more by family membership (66%, 56-75, for age at onset; 74%, 65-82, for age at death). In the GRN group, only 2% (0-10) of the variability of age at onset and 9% (3-21) of that of age of death was explained by the specific mutation, whereas 14% (9-22) of the variability of age at onset and 20% (12-30) of that of age at death was explained by family membership. In the C9orf72 group, family membership explained 17% (11-26) of the variability of age at onset and 19% (12-29) of that of age at death.
Interpretation:
Our study showed that age at symptom onset and at death of people with genetic frontotemporal dementia is influenced by genetic group and, particularly for MAPT mutations, by the specific mutation carried and by family membership. Although estimation of age at onset will be an important factor in future pre-symptomatic therapeutic trials for all three genetic groups, our study suggests that data from other members of the family will be particularly helpful only for individuals with MAPT mutations. Further work in identifying both genetic and environmental factors that modify phenotype in all groups will be important to improve such estimates.
Funding:
UK Medical Research Council, National Institute for Health Research, and Alzheimer's Society.
Introduction:
It is important to establish the natural history of familial frontotemporal lobar degeneration (f-FTLD) and provide clinical and biomarker data for planning these studies, particularly in the asymptomatic phase.
Methods:
The Longitudinal Evaluation of Familial Frontotemporal Dementia Subjects protocol was designed to enroll and follow at least 300 subjects for more than at least three annual visits who are members of kindreds with a mutation in one of the three most common f-FTLD genes-microtubule-associated protein tau, progranulin, or chromosome 9 open reading frame 72.
Results:
We present the theoretical considerations of f-FTLD and the aims/objectives of this protocol. We also describe the design and methodology for evaluating and rating subjects, in which detailed clinical and neuropsychological assessments are performed, biofluid samples are collected, and magnetic resonance imaging scans are performed using a standard protocol.
Discussion:
These data and samples, which are available to interested investigators worldwide, will facilitate planning for upcoming disease-modifying therapeutic trials in f-FTLD.
Frontotemporal dementia (FTD) is a common neurogenerative disorder characterized by neurodegeneration in the frontal and temporal lobes. Heterozygous mutations in the gene encoding progranulin (PGRN) are a common genetic cause of FTD. Recently PGRN has emerged as an important regulator of lysosomal function. Here, we examined the impact of PGRN mutations on the processing of full-length prosaposin to individual saposins, which are critical regulators of sphingolipid metabolism. Using FTD-PGRN patient-derived cortical neurons differentiated from induced pluripotent stem cells (iPSCs), as well as post-mortem tissue from patients with FTLD-PGRN, we show that PGRN haploinsufficiency results in impaired processing of prosaposin to saposin C, a critical activator of the lysosomal enzyme glucocerebrosidase (GCase). Additionally, we found that PGRN mutant neurons had reduced lysosomal glucocerebrosidase (GCase) activity, lipid accumulation and increased insoluble α- synuclein relative to isogenic controls. Importantly, this effect on GCase activity was rescued by treatment with saposin C. Together, these findings suggested that reduced GCase activity due to impaired processing of prosaposin may contribute to pathogenesis of FTD resulting from PGRN mutations.
Heterozygous loss-of-function mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal lobar degeneration with TDP-43 pathology type A (FTLD-TDP type A). PGRN is a highly conserved, secreted glycoprotein and functions in the central nervous system as a key modulator of microglial function. Hence, altered microglial function caused by PGRN deficiency may be tied to the pathogenesis of FTLD-TDP. Our previous studies showed that haploinsufficiency of GRN mutations extends to microglial PGRN expression in the hippocampal CA1 region. In this study, we found that the CA1 sector was associated with less neuronal loss and more frequent TDP-43 inclusions in FTLD-TDP type A cases with GRN mutations than in sporadic cases. In addition, the CA1 region in GRN mutation cases contained more rod-like microglia, which also had reduced PGRN expression. These findings suggest that the profile of TDP-43 inclusions, neuronal number, and microgliosis in the CA1 sector of FTLD-TDP type A cases may be influenced by GRN gene expression status.
Neuronal ceroid lipofuscinosis (NCL) is a group of progressive neurodegenerative disorders characterized by intracellular accumulation of ceroid lipopigments. Based on gene defect of NCL-associated proteins, 14 types of NCL have been described till date. NCL type 11 was first described in 2014 in two siblings as adult-onset NCL and was found to be due to a homozygous progranulin gene mutation. These siblings had progressive retinopathy, recurrent generalized seizures, moderate ataxia and subtle cognitive dysfunction. Palinopsia was present and MRI showed selective and severe cerebellar atrophy which was progressive with age. There have been no further reports of NCL 11 in literature. We here present a 14-year old girl born to second degree consanguineous couple who presented with gradually increasing frequency of seizures for the past 1 year without any signs of visual abnormalities and dementia. She had an elder sister who had progressive seizures and dementia from 8 years of age and died after few years. Her electroencephalogram showed frequent generalized epileptiform discharges and magnetic resonance imaging (MRI) showed pure cerebellar atrophy mainly affecting the vermis. MRI findings suggested a neurodegenerative disorder like NCL and prompted us to go for whole exome screen which revealed NCL type 11 due to homozygous mutation c.912G>A (p.Trp304Ter) in exon 9 of GRN gene (OMIM#614706). To the best of our knowledge this is the third case of NCL 11 and the first from Asia.
Treatment of the neuronal ceroid lipofuscinoses, also known as Batten disease, is at the start of a new era because of diagnostic and therapeutic advances relevant to this group of inherited neurodegenerative and life-limiting disorders that affect children. Diagnosis has improved with the use of comprehensive DNA-based tests that simultaneously screen for many genes. The identification of disease-causing mutations in 13 genes provides a basis for understanding the molecular mechanisms underlying neuronal ceroid lipofuscinoses, and for the development of targeted therapies. These targeted therapies include enzyme replacement therapies, gene therapies targeting the brain and the eye, cell therapies, and pharmacological drugs that could modulate defective molecular pathways. Such therapeutic developments have the potential to enable earlier diagnosis and better targeted therapeutic management. The first approved treatment is an intracerebroventricularly administered enzyme for neuronal ceroid lipofuscinosis type 2 disease that delays symptom progression. Efforts are underway to make similar progress for other forms of the disorder.
The ancient and pluripotent progranulins contain multiple repeats of a cysteine‐rich sequence motif of ∼60 amino acids, called the granulin/epithelin module (GEM) with a prototypic structure of four β‐hairpins zipped together by six inter‐hairpin disulfide bonds. Prevalence of this disulfide‐enforced structure is assessed here by an expression screening of 19 unique GEM sequences of the four progranulins in the zebrafish genome, progranulins 1, 2, A and B. While a majority of the expressed GEM peptides did not exhibit uniquely‐folded conformations, module AaE from progranulin A and AbB from progranulin B were found to fold into the protopypic 4‐hairpin structure along with disulfide formation. Module AaE has the most‐rigid three‐dimensional structure with all four β‐hairpins defined using high‐resolution (H‐¹⁵N) NMR spectroscopy, including 492 inter‐proton nuclear Overhauser effects, 23 ³J(HN,Hα) coupling constants, 22 hydrogen bonds as well as residual dipolar coupling constants. Three‐dimensional structure of AaE and the partially‐folded AbB re‐iterate the conformational stability of the N‐terminal stack of two beta‐hairpins and varying degrees of structural flexibility for the C‐terminal half of the 4‐hairpin global fold of the GEM repeat. A cell‐based assay demonstrated a functional activity for the zebrafish granulin AaE in promoting the survival of neuronal cells, similarly to what has been found for the corresponding granulin E module in human progranulin. Finally, this work highlights the remaining challenges in structure‐activity studies of proteins containing the GEM repeats, due to the apparent prevalence of structural disorder in GEM motifs despite potentially a high density of intramolecular disulfide bonds. This article is protected by copyright. All rights reserved.
Background:
Loss-of-function mutations in GRN cause frontotemporal lobar degeneration (FTLD). Patients with GRN mutations present with a uniform subtype of TAR DNA-binding protein 43 (TDP-43) pathology at autopsy (FTLD-TDP type A); however, age at onset and clinical presentation are variable, even within families. We aimed to identify potential genetic modifiers of disease onset and disease risk in GRN mutation carriers.
Methods:
The study was done in three stages: a discovery stage, a replication stage, and a meta-analysis of the discovery and replication data. In the discovery stage, genome-wide logistic and linear regression analyses were done to test the association of genetic variants with disease risk (case or control status) and age at onset in patients with a GRN mutation and controls free of neurodegenerative disorders. Suggestive loci (p<1 × 10-5) were genotyped in a replication cohort of patients and controls, followed by a meta-analysis. The effect of genome-wide significant variants at the GFRA2 locus on expression of GFRA2 was assessed using mRNA expression studies in cerebellar tissue samples from the Mayo Clinic brain bank. The effect of the GFRA2 locus on progranulin concentrations was studied using previously generated ELISA-based expression data. Co-immunoprecipitation experiments in HEK293T cells were done to test for a direct interaction between GFRA2 and progranulin.
Findings:
Individuals were enrolled in the current study between Sept 16, 2014, and Oct 5, 2017. After quality control measures, statistical analyses in the discovery stage included 382 unrelated symptomatic GRN mutation carriers and 1146 controls free of neurodegenerative disorders collected from 34 research centres located in the USA, Canada, Australia, and Europe. In the replication stage, 210 patients (67 symptomatic GRN mutation carriers and 143 patients with FTLD without GRN mutations pathologically confirmed as FTLD-TDP type A) and 1798 controls free of neurodegenerative diseases were recruited from 26 sites, 20 of which overlapped with the discovery stage. No genome-wide significant association with age at onset was identified in the discovery or replication stages, or in the meta-analysis. However, in the case-control analysis, we replicated the previously reported TMEM106B association (rs1990622 meta-analysis odds ratio [OR] 0·54, 95% CI 0·46-0·63; p=3·54 × 10-16), and identified a novel genome-wide significant locus at GFRA2 on chromosome 8p21.3 associated with disease risk (rs36196656 meta-analysis OR 1·49, 95% CI 1·30-1·71; p=1·58 × 10-8). Expression analyses showed that the risk-associated allele at rs36196656 decreased GFRA2 mRNA concentrations in cerebellar tissue (p=0·04). No effect of rs36196656 on plasma and CSF progranulin concentrations was detected by ELISA; however, co-immunoprecipitation experiments in HEK293T cells did suggest a direct binding of progranulin and GFRA2.
Interpretation:
TMEM106B-related and GFRA2-related pathways might be future targets for treatments for FTLD, but the biological interaction between progranulin and these potential disease modifiers requires further study. TMEM106B and GFRA2 might also provide opportunities to select and stratify patients for future clinical trials and, when more is known about their potential effects, to inform genetic counselling, especially for asymptomatic individuals.
Funding:
National Institute on Aging, National Institute of Neurological Disorders and Stroke, Canadian Institutes of Health Research, Italian Ministry of Health, UK National Institute for Health Research, National Health and Medical Research Council of Australia, and the French National Research Agency.
Background
Progranulin is an adipokine that is involved in the inflammatory response, glucose metabolism, insulin resistance, and may therefore be involved in chronic subclinical inflammation associated with the pathogenesis of childhood obesity. We aimed to investigate the association of circulating progranulin levels with metabolic parameters in children and to assess the importance of progranulin as a biomarker for metabolic diseases.
Methods
A total of 150 children were consecutively recruited from the Pediatric Nutrition Clinics at King Abdulaziz University Hospital in Jeddah, Saudi Arabia. Children were classified into four groups based on quartile for serum progranulin. Anthropometric variables were measured in all study subjects. Fasting blood samples were collected for measurement of blood glucose, insulin and lipid profile.
Results
Children within the upper quartile for serum progranulin concentration were heavier, more insulin resistant and had higher concentrations of serum total cholesterol, triglycerides, insulin and high sensitivity C reactive protein compared to those in the lower quartile. On correlation analysis, serum progranulin concentrations were significantly related to general and central adiposity, metabolic parameters, markers of inflammation and insulin resistance. Stepwise multiple regression showed that 26.6% of the variability in serum progranulin could be explained by measures of adiposity.
Conclusions
The increased serum progranulin concentrations were closely related to measures of adiposity, metabolic parameters, inflammatory marker and insulin resistance indices, suggesting that progranulin may be an excellent biomarker for obesity in childhood.
Neurodegenerative diseases pose an extraordinary threat to the world's aging population, yet no disease-modifying therapies are available. Although genome-wide association studies (GWASs) have identified hundreds of risk loci for neurodegeneration, the mechanisms by which these loci influence disease risk are largely unknown. Here, we investigated the association between common genetic variants at the 7p21 locus and risk of the neurodegenerative disease frontotemporal lobar degeneration. We showed that variants associated with disease risk correlate with increased expression of the 7p21 gene TMEM106B and no other genes; co-localization analyses implicated a common causal variant underlying both association with disease and association with TMEM106B expression in lymphoblastoid cell lines and human brain. Furthermore, increases in the amount of TMEM106B resulted in increases in abnormal lysosomal phenotypes and cell toxicity in both immortalized cell lines and neurons. We then combined fine-mapping, bioinformatics, and bench-based approaches to functionally characterize all candidate causal variants at this locus. This approach identified a noncoding variant, rs1990620, that differentially recruits CTCF in lymphoblastoid cell lines and human brain to influence CTCF-mediated long-range chromatin-looping interactions between multiple cis-regulatory elements, including the TMEM106B promoter. Our findings thus provide an in-depth analysis of the 7p21 locus linked by GWASs to frontotemporal lobar degeneration, nominating a causal variant and causal mechanism for allele-specific expression and disease association at this locus. Finally, we show that genetic variants associated with risk of neurodegenerative diseases beyond frontotemporal lobar degeneration are enriched in CTCF-binding sites found in brain-relevant tissues, implicating CTCF-mediated gene regulation in risk of neurodegeneration more generally.
Frontotemporal dementia (FTD) encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments. Heterozygous mutations in progranulin (PGRN) cause familial FTD and result in decreased PGRN expression, while homozygous mutations result in complete loss of PGRN expression and lead to the neurodegenerative lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL). However, how dose-dependent PGRN mutations contribute to these two different diseases is not well understood. Using iPSC-derived human cortical neurons from FTD patients harboring PGRN mutations, we demonstrate that PGRN mutant neurons exhibit decreased nuclear TDP-43 and increased insoluble TDP-43, as well as enlarged electron-dense vesicles, lipofuscin accumulation, fingerprint-like profiles and granular osmiophilic deposits, suggesting that both FTD and NCL-like pathology are present in PGRN patient neurons as compared to isogenic controls. PGRN mutant neurons also show impaired lysosomal proteolysis and decreased activity of the lysosomal enzyme cathepsin D. Furthermore, we find that PGRN interacts with cathepsin D, and that PGRN increases the activity of cathepsin D but not cathepsins B or L. Finally, we show that granulin E, a cleavage product of PGRN, is sufficient to increase cathepsin D activity. This functional relationship between PGRN and cathepsin D provides a possible explanation for overlapping NCL-like pathology observed in patients with mutations in PGRN or CTSD, the gene encoding cathepsin D. Together, our work identifies PGRN as an activator of lysosomal cathepsin D activity, and suggests that decreased cathepsin D activity due to loss of PGRN contributes to both FTD and NCL pathology in a dose-dependent manner.
Heterozygous loss-of-function mutations in GRN, the progranulin gene, which result in progranulin (PGRN) protein haploinsufficiency, are a major cause of frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). PGRN is composed of seven and a half repeats of a highly conserved granulin motif that is cleaved to produce the granulin peptides A-G and paragranulin. To better understand the role of PGRN and granulin (Grn) peptides in the pathogenesis of neurodegeneration, we evaluated PGRN/Grn in brains of patients with Alzheimer disease, FTLD-TDP type A with or without GRN mutations, and normal individuals, using a panel of monoclonal antibodies against Grn peptides A-G. In the neocortex, Grn peptide-specific immunostains were observed, for example, membranous Grn E immunopositivity in pyramidal neurons, and Grn C immunopositivity in ramified microglia. In the hippocampus, Grn immunopositivity in the CA1 and CA2 regions showed disease-specific changes in both neurons and microglia. Most interestingly, in FTLD-TDP type A with GRN mutations, there is a 60% decrease in the density of Grn-positive microglia in the hippocampal CA1, suggesting that haploinsufficiency of the GRN mutations also extends to PGRN expression in microglia. This study provides important insights into future studies of the pathogenesis and treatment of FTLD-TDP. © 2017 American Association of Neuropathologists, Inc. All rights reserved.
Progranulin (GRN) and TMEM106B are associated with several common neurodegenerative disorders including frontotemporal lobar degeneration (FTLD). A TMEM106B variant modifies GRN-associated FTLD risk. However, their functional relationship in vivo and the mechanisms underlying the risk modification remain unclear. Here, using transcriptomic and proteomic analyses with Grn−/− and Tmem106b−/− mice, we show that, while multiple lysosomal enzymes are increased in Grn−/− brain at both transcriptional and protein levels, TMEM106B deficiency causes reduction in several lysosomal enzymes. Remarkably, Tmem106b deletion from Grn−/− mice normalizes lysosomal protein levels and rescues FTLD-related behavioral abnormalities and retinal degeneration without improving lipofuscin, C1q, and microglial accumulation. Mechanistically, TMEM106B binds vacuolar-ATPase accessory protein 1 (AP1). TMEM106B deficiency reduces vacuolar-ATPase AP1 and V0 subunits, impairing lysosomal acidification and normalizing lysosomal protein levels in Grn−/− neurons. Thus, Grn and Tmem106b genes have opposite effects on lysosomal enzyme levels, and their interaction determines the extent of neurodegeneration.
The frontotemporal lobar degeneration (FTLD) protein progranulin (PGRN) is essential for proper lysosomal function. PGRN localizes in the lysosomal compartment within the cell. Prosaposin (PSAP), the precursor of lysosomal saposin activators (saposin A, B, C, D), physically interacts with PGRN. Previously, we have shown that PGRN and PSAP facilitate each other's lysosomal trafficking. Here, we report that the interaction between PSAP and PGRN requires the linker region of saposin B and C (BC linker). PSAP protein with the BC linker mutated, fails to interact with PGRN and deliver PGRN to lysosomes in the biosynthetic and endocytic pathways. On the other hand, PGRN interacts with PSAP through multiple granulin motifs. Granulin D and E bind to PSAP with similar affinity as full‐length PGRN.
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Read the Editorial Comment for this article on page 154 .
Progranulin is a cysteine-rich secreted protein with diverse pleiotropic actions and participates in several processes, such as inflammation or tumorigenesis. Progranulin was first identified as a growth factor and, recently, it was characterised as an adipokine implicated in obesity, insulin resistance and rheumatic disease. At a central level, progranulin acts as a neurotropic and neuroprotective factor and protects from neural degeneration. In this review, we summarise the most recent research advances concerning the potential role of progranulin as a therapeutic target and biomarker in cancer, neurodegenerative and inflammatory diseases.
Background.:
Progranulin (PGRN) is an important immunomodulatory factor in a variety of inflammatory diseases. However, its role in pulmonary immunity against bacterial infection remains unknown.
Methods.:
Pneumonia was induced in PGRN-deficient and normal wild-type mice using Pseudomonas aeruginosa or Staphylococcus aureus, and we assessed the effects of PGRN on survival, bacterial burden, cytokine and chemokine production, and pulmonary leukocyte recruitment after bacterial pneumonia.
Results.:
Patients with community-acquired pneumonia displayed elevated PGRN levels. Likewise, mice with Gram-negative and Gram-positive pneumonia had increased PGRN production in the lung and circulation. Progranulin deficiency led to increased bacterial growth and dissemination accompanied by enhanced lung injury and mortality in bacterial pneumonia, which was associated with impaired recruitment of macrophages and neutrophils in the lung. The reduced number of pulmonary macrophages and neutrophils observed in PGRN-deficient mice was related to a reduction of CCL2 and CXCL1 in the lungs after bacterial pneumonia. Importantly, therapeutic administration of PGRN improved mortality in severe bacterial pneumonia.
Conclusions.:
This study supports a novel role for PGRN in pulmonary immunity and suggests that treatment with PGRN may be a viable therapy for bacterial pneumonia.
Based on the recent literature and collective experience, an international consortium developed revised guidelines for the diagnosis of behavioural variant frontotemporal dementia. The validation process retrospectively reviewed clinical records and compared the sensitivity of proposed and earlier criteria in a multi-site sample of patients with pathologically verified frontotemporal lobar degeneration. According to the revised criteria, 'possible' behavioural variant frontotemporal dementia requires three of six clinically discriminating features (disinhibition, apathy/inertia, loss of sympathy/empathy, perseverative/ compulsive behaviours, hyperorality and dysexecutive neuropsychological profile). 'Probable' behavioural variant frontotemporal dementia adds functional disability and characteristic neuroimaging, while behavioural variant frontotemporal dementia 'with definite frontotemporal lobar degeneration' requires histopathological confirmation or a pathogenic mutation. Sixteen brain banks contributed cases meeting histopathological criteria for frontotemporal lobar degeneration and a clinical diagnosis of behavioural variant frontotemporal dementia, Alzheimer's disease, dementia with Lewy bodies or vascular dementia at presentation. Cases with predominant primary progressive aphasia or extra-pyramidal syndromes were excluded. In these autopsy-confirmed cases, an experienced neurologist or psychiatrist ascertained clinical features necessary for making a diagnosis according to previous and proposed criteria at presentation. Of 137 cases where features were available for both proposed and previously established criteria, 118 (86%) met 'possible' criteria, and 104 (76%) met criteria for 'probable' behavioural variant frontotemporal dementia. In contrast, 72 cases (53%) met previously established criteria for the syndrome (P 5 0.001 for comparison with 'possible' and 'probable' criteria). Patients who failed to meet revised criteria were significantly older and most had atypical presentations with marked memory impairment. In conclusion, the revised criteria for behavioural variant fronto-temporal dementia improve diagnostic accuracy compared with previously established criteria in a sample with known fronto-temporal lobar degeneration. Greater sensitivity of the proposed criteria may reflect the optimized diagnostic features, less restrictive exclusion features and a flexible structure that accommodates different initial clinical presentations. Future studies will be needed to establish the reliability and specificity of these revised diagnostic guidelines.